Oxalic acid and its salt are produced during the normal metabolism of mammals, including human beings. Although a healthy mammal breaks down or excretes sufficient oxalate to avoid excessive accumulation of oxalate in the tissues, a number of disease states are known to be associated with malfunctions of oxalate metabolism. Firstly, about 200,000 people have to be hospitalized each year in the United States for the treatment of kidney stones and about 70% of these stones are comprised wholly or partially of oxalic acid. A much smaller number of people are afflicted with primary hyperoxaluria, a genetic metabolic disorder in which oxalate is deposited in the kidneys. Nephrolithiasis and nephrocalcinosis are usually present in patients suffering from this condition before the age of 5 and 80% of the patients die before reaching the age of 20. Secondary hyperoxaluria, having similar effects but not due to a genetic disorder, has been reported following ileal resection and jejunoileal shunt procedures, and in patients suffering from Chron's disease, diabetes mellitus, cirrhosis, pyridoxine deficiency and sarcoidosis, or in patients who have undergone methoxyfluothane anesthesia. The same complications occur in the small number of humans, and probably much larger number of household pets, who are accidentally poisoned with ethylene glycol, a common constituent of automobile radiator antifreeze. Although ethylene glycol itself is not poisonous, it is rapidly oxidized within the mammalian body to a number of oxidation products including the highly-poisonous oxalic acid. Finally, excessive oxalate excretion is frequently a complication in patients suffering from steatorrhea.
In view of the variety of clinical conditions known to be associated with malfunctions in oxalate metabolism, physicians and other medical personnel frequently require a reliable and accurate method for the measurement of oxalate in body fluids. Such oxalate assays are normally made on urine, because, in many of the aforementioned conditions, oxalate levels in blood serum are within normal ranges. The mere detection of oxalate crystals in urine is of little or no diagnostic significance, since patients with normal oxalate metabolism may also produce such crystals in the urine, as described in:
Howanitz, P. J. and Howanitz, J. H., in Todd-Sanford-Davidsohn-Clinical Diagnosis and Management by Laboratory Methods, J. B. Henry, W. B. Saunders Co., Philadelphia, Pa., 16th Edn.; and PA1 Hodgkinson, A., Oxalic Acid in Biology and Medicine, Academic Press, New York, N.Y., 1978. PA1 Hodgkinson, A. Determination of Oxalic acid in Biological Material, Clin. Chem. 16 (7), 547-557 (1970). PA1 Costello, J. Hatch, M. and Bourke, E., An enzymic method for the spectrophotometric determination of oxalic acid, J. Lab. Clin. Med. 87(5), 903-908 (1976); and PA1 Yriberri, J. and Posten, LS., A semi-automatic enzymic method for estimating urinary oxalate, Clin. Chem. 26(7), 881-884 (1980). PA1 Datta, P. K. and Meeuse, B. J. D., Moss oxalic acid oxidase-a flavoprotein. Biochim. Biophys. Acta. 17, 602(1955); PA1 Chiriboga, J., Some properties of an oxalic oxidase purified from barley seedlings, Biochem. Biophys. Res. Commun. 11, 277-282 (1963); and PA1 Chiriboga, J. Purification and properties of oxalic acid oxidase. Arch. Biochem. Biphys., 116, 516-523, (1966).
Most prior art methods for measuring oxalate in urine and other body fluids require the isolation of the oxalate by a precipitation, solvent extraction or ion-exchange absorption; see, for example:
Following the isolation of the oxalate, quantitative assay thereof is completed by colorimetry, fluorometry, gas-liquid chromatography or isotope dilution techniques. Because many of the oxalate isolation techniques used in these analytical methods are not quantitative, it is normally necessary to correct for the low recovery of oxalate by adding a .sup.14 C-labeled oxalic acid internal standard, which further complicates the analytical method. All these methods are laborious, and consequently expensive because of the amount of skilled laboratory technician time which must be employed, and none of them are suited to the use of automatic analyzers.
More recently, enzymatic methods for measuring oxalate have been developed using either oxalate decarboxylase (EC 4.1.1.2. according to the enzyme nomenclature system of the International Union of Biochemists) or oxalate oxidase (EC 1.2.3.4) as the enzyme. Oxalate decarboxylase converts oxalate to carbon dioxide and formate, and the resultant carbon dioxide can be measured manometrically, by the pH change in a carbon dioxide trapping buffer or the color change in a pH indicator buffer. However, whatever method of carbon dioxide assay is adopted, the time required for diffusion and equilibration of carbon dioxide is much longer than is desirable for a rapid analytical method.
Alternatively, the formate can be assayed with formate dehydrogenase (EC 1.2.1.2.) in an NAD/NADH coupled reaction, as described in:
This method is cumbersome and time-consuming because oxalate decarboxylase and formate dehydrogenase differ in optimum pH so that a pH adjustment is necessary during the analysis.
Oxalate oxidases produce two moles of carbon dioxide and one mole of hydrogen peroxide from each mole of oxalate. The oxalate oxidases used in prior art analytical methods have been obtained from moss and barley seedlings, as described in:
The hydrogen peroxide produced by the action of the oxalate on oxalate may be quantitatively determined by reacting it with the chromogen 3-methyl-2-benzothiazolinone (MBTH) and N,N-dimethylaniline (DMA) in the presence of a peroxidase, such as horseradish peroxidase.
The analytical methods based upon oxalate oxidase from moss and barley seedlings are probably the most convenient oxalate assay methods discovered to date, but they are still subject to two major disadvantages. Firstly, the raw material must be specially grown or prepared prior to the isolation of the oxalate oxidase. The mosses used only grow wild (it is apparently not economical to cultivate them commercially for this purpose) and the supply of moss is thus subject to all the vagaries associated with the gathering of a wild product. Although barley is of course readily available, barley seedlings are not a commercial product and the barley must be germinated especially for producing the enzyme. In view of the relatively low yield of enzyme from the moss and barley seedlings, the gathering or production of the raw material represents a considerable problem. Secondly, the oxalate oxidases from moss and barley seedlings are strongly inhibited by sodium, chloride and other common water-soluble ions which are normally present in body fluids, especially urine. Thus, if such oxalate oxidase analytical methods are to be used for the analysis of oxalate in body fluid, either substantial errors due to the presence of the interfering ions must be accepted, or elaborate pretreatment of the body fluid is necessary to remove the interfering ions before the body fluids are analyzed.
Thus, there is at present no simple, inexpensive and accurate method of assaying oxalate in body fluids. This need is well-recognized by those skilled in the art; for example, despite the plethora of oxalate assay methods described in the literature, the National Institutes of Health a few years ago awarded three contracts for the development of rapid and accurate oxalate assays. It is widely believed that the number of oxalate assays requested by physicians is considerably reduced because the existing assays are so time-consuming, costly and unreliable in their results, and that if a simple, quick and inexpensive oxalate assay method could be provided, far more oxalate tests would be performed, with consequent improvement in the diagnosis and treatment of conditions related to malfunction of oxalate metabolism.
Accordingly, this invention seeks to provide a relatively quick simple and inexpensive method for the assay of oxalate, especially in biological fluids.